Tyrosine Phosphorylation on Spleen Tyrosine Kinase (Syk) Is Differentially Regulated in Human and Murine Platelets by Protein Kinase C Isoforms

Protein kinase C (PKC) isoforms differentially regulate platelet functional responses downstream of glycoprotein VI (GPVI) signaling, but the role of PKCs regulating upstream effectors such as Syk is not known. We investigated the role of PKC on Syk tyrosine phosphorylation using the pan-PKC inhibitor GF109203X (GFX). GPVI-mediated phosphorylation on Syk Tyr-323, Tyr-352, and Tyr-525/526 was rapidly dephosphorylated, but GFX treatment inhibited this dephosphorylation on Tyr-525/526 in human platelets but not in wild type murine platelets. GFX treatment did not affect tyrosine phosphorylation on FcRγ chain or Src family kinases. Phosphorylation of Lat Tyr-191 and PLCγ2 Tyr-759 was also increased upon treatment with GFX. We evaluated whether secreted ADP is required for such dephosphorylation. Exogenous addition of ADP to GFX-treated platelets did not affect tyrosine phosphorylation on Syk. FcγRIIA- or CLEC-2-mediated Syk tyrosine phosphorylation was also potentiated with GFX in human platelets. Because potentiation of Syk phosphorylation is not observed in murine platelets, PKC-deficient mice cannot be used to identify the PKC isoform regulating Syk phosphorylation. We therefore used selective inhibitors of PKC isoforms. Only PKCβ inhibition resulted in Syk hyperphosphorylation similar to that in platelets treated with GFX. This result indicates that PKCβ is the isoform responsible for Syk negative regulation in human platelets. In conclusion, we have elucidated a novel pathway of Syk regulation by PKCβ in human platelets.     

Disruption of Cell–Substrate Adhesion Activates the Protein Tyrosine Kinase pp60

Treatment of confluent chicken embryo fibroblasts (CEFs) with trypsin results in a dose- and time-dependent increase in c-Src protein tyrosine kinase (PTK) activity. A similar, but less marked, increase in c-Src PTK activity occurs upon incubation of CEFs in calcium-free phosphate-buffered saline, which also causes a decrease in cell–substrate adhesion. The increase in c-Src PTK activity following disruption of cell–substrate adhesion correlates with a decrease in the phosphorylation of c-Src at the regulatory site, Tyr527. The phosphotyrosine phosphatase inhibitor phenylarsine oxide blocks the increase in c-Src PTK activity seen following treatment with trypsin and the morphological changes associated with the disruption of cell–substrate adhesion. In contrast, disruption of cell–substrate adhesion causes a decrease in FAK PTK activity that rapidly returns to control levels when the cells are plated on fibronection-coated dishes. Treatment of cells with cytochalasin D, which disrupts actin filaments but not cell–substrate adhesion, causes only a slight increase in c-Src PTK activity. Thus, these studies demonstrate a ligand-independent mechanism for the activation of c-Src that is consistent with its role in both cell adhesion and cell motility. Furthermore, these data suggest that similar to adhesion, loss of adhesion is not a passive process but can activate specific signaling pathways that may have significant effects on cellular function.     

Ca2+/Calmodulin and Apo-Calmodulin Both Bind to and Enhance the Tyrosine Kinase Activity of c-Src

Src family non-receptor tyrosine kinases play a prominent role in multiple cellular processes, including: cell proliferation, differentiation, cell survival, stress response, and cell adhesion and migration, among others. And when deregulated by mutations, overexpression, and/or the arrival of faulty incoming signals, its hyperactivity contributes to the development of hematological and solid tumors. c-Src is a prototypical member of this family of kinases, which is highly regulated by a set of phosphorylation events. Other factor contributing to the regulation of Src activity appears to be mediated by the Ca²⁺ signal generated in cells by different effectors, where the Ca²⁺-receptor protein calmodulin (CaM) plays a key role. In this report we demonstrate that CaM directly interacts with Src in both Ca²⁺-dependent and Ca²⁺-independent manners in vitro and in living cells, and that the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibits the activation of this kinase induced by the upstream activation of the epidermal growth factor receptor (EGFR), in human carcinoma epidermoide A431 cells, and by hydrogen peroxide-induced oxidative stress, in both A431 cells and human breast adenocarcinoma SK-BR-3 cells. Furthermore, we show that the Ca²⁺/CaM complex strongly activates the auto-phosphorylation and tyrosine kinase activity of c-Src toward exogenous substrates, but most relevantly and for the first time, we demonstrate that Ca²⁺-free CaM (apo-CaM) exerts a far higher activatory action on Src auto-phosphorylation and kinase activity toward exogenous substrates than the one exerted by the Ca²⁺/CaM complex. This suggests that a transient increase in the cytosolic concentration of free Ca²⁺ is not an absolute requirement for CaM-mediated activation of Src in living cells, and that a direct regulation of Src by apo-CaM could be inferred.     

c-Abl蛋白酪氨酸激酶形成同源二聚体

c-Abl是非受体酪氨酸激酶,它在细胞内被一些基因毒性的、氧化的及其它形式的压力所激活。目前研究证明：应用标记的c-Abl发现其在细胞内可以相互形成同源二聚体,并且一分子c-Abl的N末端区域与相应的另一分子的C末端相互作用形成二聚体。实验进一步表明： cAbl SH3 结构域结合到另一c-Abl 分子富含脯氨酸的C-末端约958-982氨基酸区域。如果去除c-Abl 富含脯氨酸的结构域,就会阻止二聚体的形成。这些结果首先证实了c-Abl在细胞内可以相互形成同源二聚体,并暗示着二聚体的形成可能影响着c-Abl活性的调节。     

Liver Kinase B1 Expression Promotes Phosphatase Activity and Abrogation of Receptor Tyrosine Kinase Phosphorylation in Human Cancer Cells

Aberrant receptor tyrosine kinase phosphorylation (pRTK) has been associated with diverse pathological conditions, including human neoplasms. In lung cancer, frequent liver kinase B1 (LKB1) mutations correlate with tumor progression, but potential links with pRTK remain unknown. Heightened and sustained receptor activation was demonstrated by LKB1-deficient A549 (lung) and HeLaS3 (cervical) cancer cell lines. Depletion (siRNA) of endogenous LKB1 expression in H1792 lung cancer cells also correlated with increased pRTK. However, ectopic LKB1 expression in A549 and HeLaS3 cell lines, as well as H1975 activating-EGF receptor mutant lung cancer cell resulted in dephosphorylation of several tumor-enhancing RTKs, including EGF receptor, ErbB2, hepatocyte growth factor receptor (c-Met), EphA2, rearranged during transfection (RET), and insulin-like growth factor I receptor. Receptor abrogation correlated with attenuation of phospho-Akt and increased apoptosis. Global phosphatase inhibition by orthovanadate or depletion of protein tyrosine phosphatases (PTPs) resulted in the recovery of receptor phosphorylation. Specifically, the activity of SHP-2, PTP-1β, and PTP-PEST was enhanced by LKB1-expressing cells. Our findings provide novel insight on how LKB1 loss of expression or function promotes aberrant RTK signaling and rapid growth of cancer cells.     

Regulation of the Protein Kinase Activity of the Human Insulin Receptor

Abstract

The insulin receptor is a hormone-dependent protein tyrosine kinase that belongs to the family of tyrosine kinases associated with growth factor receptors and oncogene products. The activity of the insulin receptor kinase is regulated by the phosphorylation state of specific domains of the protein. Phosphorylation of the receptor on tyrosine residues activates its kinase activity whereas phosphorylation on serine and/or threonine residues inhibits it. In this review, we discuss the evidence that supports a role of the kinase activity of the receptor in the molecular mechanism of insulin action.     

小肠平滑肌胰岛素受体的特征及其蛋白激酶活性

 本实验对狗小肠平滑肌中胰岛素受体的结构和特征进行了分析研究。通过麦胚凝集素琼脂糖和两次Sepharose-CL-6B凝胶层析从平滑肌中纯化胰岛素受体,达到电泳纯。SDS-聚丙烯酰胺凝胶电泳证明胰岛素受体是由两个亚基组成的,分子量分别为135kD和90kD。磷酸化实验证明平滑肌胰岛素受体具有胰岛素依赖性蛋白激酶活性,能催化自身的β亚基磷酸化和底物的磷酸化。Scatchard分析表明胰岛素和受体的结合呈(?)协同效应,最大结合率为13μg胰岛素/mg蛋白质。     

Antibacterial Activity of the Purified Peroxidase from Human Parotid Saliva

The peroxidase of human parotid saliva has been purified by concentration, gel filtration on Sephadex G-200, and ion exchange chromatography on Amberlite CG-50. The purified product was devoid of amylase activity, lysozyme activity, and immunoglobulin A (IgA). However, it had an inhibitory effect on the growth of Lactobacillus acidophilus in complete growth medium and on lysine accumulation by L. acidophilus in a buffer-glucose medium, when combined with thiocyanate ions. The concentrations of peroxidase and thiocyanate ions employed were within the range found in saliva. The fractions which contained IgA did not have an anti-bacterial effect on L. acidophilus under the conditions employed. Parotid saliva also contained low molecular weight inhibitors of peroxidase activity. These studies support the involvement of the salivary peroxidase in an antibacterial system in saliva.     

甲壳胺对HepG2细胞蛋白酪氨酸激酶和磷酸酶活性的影响

目的:初步探讨甲壳胺诱导人肝癌Hep G2细胞凋亡的信号转导机制。方法:采用酶联免疫法,动态检测甲壳胺作用于Hep G2细胞后,细胞膜相及胞浆内的蛋白酪氨酸激酶(PTK)及蛋白酪氨酸磷酸酶(PTP)活性的变化。结果:甲壳胺可以抑制Hep G2细胞内的PTK活性,并呈一定的浓度依赖性;甲壳胺作用Hep G2细胞后,随着PTK活性的减弱,PTP的活性也短暂下降。结论:甲壳胺诱导Hep G2细胞凋亡时,涉及到PTK的活性改变。观察到膜相蛋白中PTK的活性改变早于胞浆蛋白,提示可能存在一个信号的跨膜转运过程;同时伴有PTP的活性变化,可能反映了胞内蛋白酪氨酸残基的磷酸化与去磷酸化即时调节机制。     

Chemical Constituents from the Heartwood of Haematoxylon campechianum as Protein Tyrosine Kinase Inhibitors

In a previous study, we showed that a series of homoisoflavonoids from the stems of Haematoxylon campechianum possess potent protein tyrosine kinase inhibitory activity. In a further chemical investigation of the heartwood of H. campechianum, three new homoisoflavonoids, epihematoxylol B ( 2 ), 10‐O‐methylhematoxylol B ( 3 ), and 10‐O‐methylepihematoxylol B ( 4 ), were isolated and identified, together with 15 known compounds, including three homoisoflavonoids, three flavonoids, six lignans, and three unsaturated fatty acids. The structures of the new compounds were established on the basis of 1D‐ and 2D‐NMR and other spectroscopic analyses.     

Protein Kinase C Regulates the Cell Surface Activity of Endothelin-Converting Enzyme-1

The potent vasoconstrictor endothelin is a 21 amino acid peptide whose principal physiological function is to regulate vascular tone. The generation of endothelin is crucially dependent on the local presence and activity of endothelin converting enzyme-1 (ECE-1) expressed on the surface of vascular endothelial cells. In this study, we have shown in endothelial cells that the enzyme is phosphorylated, and that phosphorylation is increased by phorbol ester stimulation of protein kinase C (PKC). Furthermore, by monitoring specific ECE-1 activity on the surface of live cells, we also show that following PKC activation, enzyme activity is significantly increased at the cell surface, where it is positioned to catalyse the generation of active endothelin. We believe this novel finding is unprecedented for a peptide processing enzyme. Indeed, this new knowledge regarding the control of endothelin production by regulating ECE-1 activity at the cell surface opens up a new area of endothelin biology and will provide novel insights into the physiology and pathophysiology of endothelin and endothelin-associated diseases. In addition, the information generated in these studies may provide valuable new insights into potential extra- and intracellular targets for the pharmacological and perhaps even therapeutic regulation of endothelin production and thus vascular tone.Australian Peptide Conference Issue.     

Involvement of Protein Kinase D1 in Signal Transduction from the Protein Kinase C Pathway to the Tyrosine Kinase Pathway in Response to Gonadotropin-releasing Hormone

The receptor for gonadotropin-releasing hormone (GnRH) belongs to the G protein-coupled receptors (GPCRs), and its stimulation activates extracellular signal-regulated protein kinase (ERK). We found that the transactivation of ErbB4 was involved in GnRH-induced ERK activation in immortalized GnRH neurons (GT1–7 cells). We found also that GnRH induced the cleavage of ErbB4. In the present study, we examined signal transduction for the activation of ERK and the cleavage of ErbB4 after GnRH treatment. Both ERK activation and ErbB4 cleavage were completely inhibited by YM-254890, an inhibitor of G_q/11 proteins. Down-regulation of protein kinase C (PKC) markedly decreased both ERK activation and ErbB4 cleavage. Experiments with two types of PKC inhibitors, Gö 6976 and bisindolylmaleimide I, indicated that novel PKC isoforms but not conventional PKC isoforms were involved in ERK activation and ErbB4 cleavage. Our experiments indicated that the novel PKC isoforms activated protein kinase D (PKD) after GnRH treatment. Knockdown and inhibitor experiments suggested that PKD1 stimulated the phosphorylation of Pyk2 by constitutively activated Src and Fyn for ERK activation. Taken together, it is highly possible that PKD1 plays a critical role in signal transduction from the PKC pathway to the tyrosine kinase pathway. Activation of the tyrosine kinase pathway may be involved in the progression of cancer.     

AMPK上游激酶——肿瘤抑制因子LKB1

AMPK是哺乳动物细胞中高度保守的蛋白质,是细胞的“代谢感受器”。AMPK的活化需要上游激酶(AMPKK)对AMPKα亚基活化环上Thr172进行磷酸化来完成。最近的研究发现,肿瘤抑制因子LKB1可以磷酸化Thr172进而激活AMPK,因此认为它是AMPKK家族的成员。     

Fibroblast Growth Factors Stimulate Protein Tyrosine Phosphorylation and Mitogen-Activated Protein Kinase Activity in Primary Cultures of Hippocampal Neurons

Abstract: Fibroblast growth factors (FGFs) are not only mitogens, but they also promote the differentiation of various cell types. For instance, basic FGF (bFGF) provides a critical trophic support for hippocampal neurons in culture. To elicit their biological effects, FGFs interact with high-affinity receptors that are transmembrane proteins with a cytoplasmic portion containing a tyrosine kinase activity. The tyrosine phosphorylation pattern was examined in primary cultures of hippocampal neurons derived from rat embryos. In these cultures grown for 3 days in the absence of serum, the addition of bFGF causes a rapid increase of tyrosine phosphorylation for various proteins with an optimal level after 5 min of bFGF exposure. Concomitantly, bFGF activates mitogen-activated protein kinase (MAP kinase) activity measured with a selective MAP kinase peptide. The activity increased rapidly after the addition of bFGF and remained elevated even when cultures were treated for 1 h with bFGF. Both acidic and basic FGF were able to enhance protein tyrosine phosphorylation and MAP kinase activity, whereas nerve growth factor and epidermal growth factor did not elicit any of these responses. These data indicate that some of the transduction signals (i.e., tyrosine phosphorylation and activation of MAP kinase) that have been described for the proliferative effect of FGFs are also involved when FGFs act as trophic factors for postmitotic neurons in culture.     

阿勒泰黄芪提取物对蛋白酪氨酸磷酸酯酶1B的抑制作用

本文探讨了阿勒泰黄芪不同提取物对蛋白酪氨酸磷酸酯酶1B(PTP1B)的抑制作用.采用分光光度法测定了提取物中的黄酮和皂苷含量;通过体外酶促动力学方法检测了不同提取物对PTP1B的影响,并确定了抑制类型;并采用氧化酶法检测了阿勒泰黄芪提取物对细胞利用葡萄糖能力的作用.结果表明,阿勒泰黄芪8种提取物(E1 ～8)中黄酮含量分别为5.09、10.46、3.58、3.23、53.91、21.77、5.76和7.49 mg/mL,其中E1、E2、E6、E7、E8皂苷含量分别为16.53、27.45、21.90、10.21和8.96 mg/mL;各提取物对PTP1B活性均表现出抑制作用,其中E1、E2、E7、E8的IC50分别为34.8、4.7、7.35和7.15 μg/mL,E1、E7和E8是竞争性抑制,E2是混合型竞争性抑制.E1、E2、E5、E7和E8较明显的提高了CHO-K1细胞对葡萄糖的利用.提示皂苷可能是阿勒泰黄芪抑制PTP1B活性的主要物质,通过PTP1B途径有效了提高细胞利用葡萄糖的能力.本研究为阿勒泰黄芪开发为防治糖尿病及改善胰岛素抵抗的药物或保健品提供实验依据.     

三种维药提取物对蛋白酪氨酸磷酸酶1B抑制作用的研究

蛋白酪氨酸磷酸酶1B(PTP1B)是胰岛索增敏的新靶点之一.本文研究了芳香新塔花、沙生蜡菊、驱虫斑鸠菊等3种维药石油醚提取物对PTP1B活性的影响及其对酶的抑制类型.结果表明,所构建的原核表达系统能高表达重组PTP1B(his-PTP1B1-321),分子量为40.8 kDa.3种维药提取物对PTP1B均表现出不同程度...     

Activation of Protein Kinase C by Purified Bovine Brain 14-3-3: Comparison with Tyrosine Hydroxylase Activation

Abstract: In the course of the purification of 14-3-3 protein (14-3-3) we found that 14-3-3 isolated from bovine forebrain activates protein kinase C (PKC), rather than the previously reported protein kinase C inhibitory activity (KCIP). We have characterized the 14-3-3 activation of PKC. The physical properties of purified PKC activator are the same as those previously reported for 14-3-3 and KCIP; i.e., (1) it is composed of subunits of molecular weight 32,000, 30,000, and 29,000; (2) it is homogeneous with respect to molecular weight, as judged by native gradient-gel electrophoresis, with a molecular weight of 53,000; and (3) it is composed of at least six isoforms when analyzed by reverse-phase HPLC. The concentration dependence of PKC activation by 14-3-3 is in the same range as that shown previously for KCIP inhibition of PKC, and as that required for 14-3-3 activation of tyrosine hydroxylase; a maximal stimulation of two- to three-fold occurs at 40–100 µg/ml. 14-3-3's activation of PKC is sensitive to α-chymotrypsin digestion but is not heat labile. Activation is specific to PKC; at least two other protein kinases, cyclic AMP- and calcium/calmodulin-dependent protein kinases, are not activated. The activation of PKC by 14-3-3 is independent of phosphatidylserine and calcium and, as such, is an alternative mechanism for the activation of PKC that obviates its translocation to membranes.     

Neurite Outgrowth Stimulated by the Tyrosine Kinase Inhibitor Herbimycin A Requires Activation of Tyrosine Kinases and Protein Kinase C

Abstract: Activation of tyrosine kinases is established as an important mechanism for controlling growth cone motility and neurite outgrowth. We have tested the effects of a range of tyrosine kinase inhibitors on neurite outgrowth from postnatal day 4 cerebellar granule cells cultured over confluent monolayers of 3T3 fibroblasts. The only agent that had any effect was herbimycin A, which stimulated neurite outgrowth. The response is shown to be attributable to a direct effect of this tyrosine kinase inhibitor on neurones. The neurite outgrowth response to herbimycin A was inhibited by two other tyrosine kinase inhibitors, which on their own did not affect neurite outgrowth. The data suggest that the response to herbimycin A reflects either a direct or indirect activation of one or more protein tyrosine kinases. Independent signalling events downstream from tyrosine kinase activation underlying the neurite outgrowth response to herbimycin A include increased activity of protein kinase C and calcium influx into neurones through both N-and L-type calcium channels.     

GAK is phosphorylated by c-Src and translocated from the centrosome to chromatin at the end of telophase

Cyclin G-associated kinase (GAK) harbors a consensus phosphorylation motif (Y412) for c-Src; however, its physiological significance remains elusive. Here, we show that GAK is phosphorylated by c-Src not only at Y412 but also at Y1149. An anti-GAK-pY412 antibody recognized the shifted band of GAK during M phase. Immunofluorescence (IF) showed that GAK-pY412/pY1149 signals were present in the nucleus during interphase, translocated to chromosomes at prophase and prometaphase, moved to centrosomes at metaphase, and finally translocated to chromosomes at the end of telophase, when nuclear membrane formation was almost complete. These subcellular movements of GAK resemble those of DNA licensing factors. Indeed, mass spectrometry identified mini-chromosome maintenance (MCM) 3, an essential component of the DNA licensing system, as one of the association partners of GAK; immunoprecipitation-mediated Western blotting confirmed their association in vivo. These results suggest that the c-Src_GAK_MCM axis plays an important role in cell cycle progression through control of the DNA replication licensing system.